Immunostimulatory polynucleotide/immunomodulatory molecule conjugates

ABSTRACT

Immunostimulatory polynucleotide-immunomodulatory molecule conjugate compositions are disclosed. These compositions include a polynucleotide that is linked to an immunomodulatory molecule, which molecule comprises an antigen and may further comprise immunomodulators such as cytokines and adjuvants. The polynucleotide portion of the conjugate includes at least one immunostimulatory oligonucleotide nucleotide sequence (ISS). Methods of modulating an immune response upon administration of the polynucleotide-immunomodulatory conjugate preparation to a vertebrate host are also disclosed.

RELATED U.S. PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/308,036, filed Feb. 16, 2000 now U.S. Pat. No. 6,610,661, which is anational phase filing under 35 U.S.C. §371 of PCT/US97/19004, filed Oct.9, 1997, which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 60/028,118, filed Oct. 11, 1996, each of whichapplications is incorporated herein by reference in their entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

Support for the research disclosed herein may have been provided by theNational Institutes of Health under Grant Nos. AI37305 and/or AR25443.

FIELD OF THE INVENTION

The invention relates to compositions comprising an immunomodulatorymolecule (IMM) including an antigen, conjugated to a polynucleotide thatcontains or consists of at least one immunostimulatory oligonucleotide(ISS-PN). It also relates to methods for modulating the immune responseof a vertebrate host to an antigen.

HISTORY OF THE RELATED ART

Conventionally, immunization of a host against an antigen isaccomplished by repeatedly vaccinating the host with the antigen. Whilemost current vaccines elicit reasonable antibody responses, cellularresponses (in particular, major histocompatibility complex (MHC) classI-restricted cytotoxic T cells) are generally absent or weak. For manyinfectious diseases, such as tuberculosis and malaria, humoral responsesare of little protective value against infection.

Given the weak cellular immune response to protein antigens, modulationof the immune responses to these antigens has clear importance. Theability to modify immune responses to protein or peptide antigen hasimplications for tumor therapy, for the treatment of allergic disordersand for treatment of other conditions achievable through induction of avigorous cellular immune response.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising an ISS-PN whichis conjugated to an IMM (which includes an antigen) to form ISS-PN/IMMconjugates. The ISS-PN/IMM conjugates of the invention are biologicalresponse modifiers in the sense that they modify the humoral andcellular immune response of a host to an antigen.

Specifically, the ISS-PN and IMM components of the ISS-PN/IMM conjugatessynergistically boost the magnitude of the host immune response againstan antigen to a level greater than the host immune response to eitherthe IMM, antigen or ISS-PN alone. The ISS-PN/IMM conjugates also shiftthe host cellular immune response away from the helper T lymphocyte type2 (Th2) phenotype toward a helper T lymphocyte type 1 (Th1) phenotype.These responses to ISS-PN/IMM conjugates are particularly acute duringthe important early phase of the host immune response to an antigen.

To these ends, ISS-PN/IMM conjugates are delivered by any route throughwhich antigen-sensitized host tissues will be contacted with theISS-PN/IMM conjugate. ISS-PN/IMM conjugates administered in this fashionboost both humoral (antibody) and cellular (Th1 type) immune responsesof the host. Thus, use of the method to boost the immune responsivenessof a host to subsequent challenge by a sensitizing antigen withoutimmunization avoids the risk of Th2-mediated, immunization-inducedanaphylaxis by suppressing IgE production in response to the antigenchallenge. An especially advantageous use for this aspect of theinvention is treatment of localized allergic responses in target tissueswhere the allergens enter the body, such as the skin and mucosa.

Suppression of the Th2 phenotype according to the invention is also auseful in reducing antigen-stimulated IL-4 and IL-5 production. Thus,the invention encompasses delivery of ISS-PN/IMM conjugates to a host tosuppress the Th2 phenotype associated with conventional antigenimmunization (e.g., for vaccination or allergy immunotherapy).

The shift to a Th1 phenotype achieved according to the invention isaccompanied by increased secretion of IFN α, β and γ, as well as IL-12and IL-18. Each of these cytokines enhance the host's immune defensesagainst intracellular pathogens, such as viruses. Thus, the inventionencompasses delivery of ISS-PN/IMM conjugates to a host to combatpathogenic infection.

Angiogenesis is also enhanced in the Th1 phenotype (ostensibly throughstimulation by IL-12). Thus, the invention encompasses delivery ofISS-PN/IMM conjugates to a host to stimulate therapeutic angiogenesis totreat conditions in which localized blood flow plays a significantetiological role; e.g., retinopathies.

The ISS-PN/IMM conjugates of the invention comprise an IMM conjugated toa polynucleotide that includes, or consists of, at least oneimmunostimulatory oligonucleotide (ISS-ODN) moiety. The ISS-ODN moietyis a single- or double-stranded DNA or RNA oligonucleotide having atleast 6 nucleotide bases which may include, or consist of, a modifiedoligonucleoside or a sequence of modified nucleosides.

The ISS-ODN moieties comprise, or may be flanked by, a CpG containingnucleotide sequence or a p(IC) nucleotide sequence, which may bepalindromic. Where the oligonucleotide moiety comprises a CpG sequence,it may include a hexamer structure consisting of: 5′-Purine, Purine, CG,Pyrimidine, Pyrimidine-3′. Examples of such hexamer structures areAACGTT, AGCGTT, GACGTT, GGCGTT, AACGTC, and AGCGTC.

In one aspect of the invention, the ISS-PN consists of an ISS-ODN.Alternatively, the ISS-PN comprises an ISS-ODN.

Conjugates of the invention also include PN/IMM wherein the PN serves asa carrier to introduce the IMM antigen into MHC Class I processingpathways not normally stimulated by soluble antigen, but lacks ISSactivity and therefore does not stimulate a Th1 phenotype immuneresponse. Examples of such PN/IMM are those wherein the CpG motif ismutated, for example, to a GpG motif.

In one aspect of the invention, the IMM conjugate partner to the ISS-PNconsists of an antigen. Such antigens are selected from the group ofantigens consisting of proteins, peptides, glycoproteins,polysaccharides and gangliosides.

In another aspect of the invention, the IMM conjugate partner comprisesan antigen and further comprises an immunostimulatory molecule selectedfrom the group of such molecules consisting of adjuvants, hormones,growth factors, cytokines, chemokines, targeting protein ligands, andtrans-activating factors.

In another aspect of the invention, the ISS-PN/IMM conjugate is modifiedfor targeted delivery by, for example, attachment to a monoclonalantibody, receptor ligand and/or liposome.

Pharmaceutically acceptable compositions of ISS-PN/IMM conjugates areprovided for use in practicing the methods of the invention. Whereappropriate to the contemplated course of therapy, the ISS-PN/IMMconjugates may be administered with anti-inflammatory orimmunotherapeutic agents. Thus, a particularly useful composition foruse in practicing the method of the invention is one in which ananti-inflammatory agent (e.g., a glucocorticoid) is mixed with, orfurther conjugated to, an ISS-PN/IMM conjugate.

The ISS-PN/IMM conjugates can also be provided in the form of a kitcomprising ISS-PN/IMM conjugates and any additional medicaments, as wellas a device for delivery of the ISS-PN/IMM conjugates to a host tissueand reagents for determining the biological effect of the ISS-PN/IMMconjugates on a treated host.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of data demonstrating the vigorous Th1-type immuneresponse (as measured by production of IgG2a against an IMM antigen)stimulated by ISS-PN/IMM (1:5 ratio) in comparison to the levels ofTh2-like responses stimulated by an ISS containing, antigen encodingplasmid (pACB-Z); the antigen alone (β-gal); the antigen mixed with anISS (1:5 ratio); the antigen conjugated to a non-stimulatory PN (mISSconj; 1:5 ratio); the antigen in adjuvant (alum) and, for reference, theIgG2a levels in naive (unexposed) mice. The horizontal axis representsthe levels (units/ml) of antibody; the vertical axis represents thenumber of weeks following primary antigen exposure.

FIG. 2 is a graph of data demonstrating the levels of Th2-type immuneresponses (as measured by production of IgG1 against an IMM antigen)stimulated by an ISS containing, antigen encoding plasmid (pACB-Z); theantigen alone (β-gal); the antigen mixed with an ISS (1:5 ratio); theantigen conjugated to a non-stimulatory PN (mISS conj; 1:5 ratio); theantigen in adjuvant (alum) and, for reference, the IgG1 levels in naive(unexposed) mice, all as compared to the vigorous Th1-type immuneresponse produced in mice immunized with ISS-PN/IMM (1:5 ratio). Thehorizontal axis represents the levels (units/ml) of antibody; thevertical axis represents the number of weeks following primary antigenexposure.

FIG. 3 is a graph of data demonstrating the vigorous Th1-type immuneresponse (as measured by production of IgG2a against an IMM antigen)stimulated by ISS-PN/IMM in comparison to the levels of Th2-likeresponses stimulated by the antigen alone (AgE) and antigen conjugatedto a non-stimulatory PN (mISS conj). Antigen to PN ratios are all 1:5.The horizontal axis represents the levels (units/ml) of antibody; thevertical axis shows the levels at 4 weeks following primary antigenexposure (shaded bars) and at 2 weeks following secondary antigenchallenge (solid bars).

FIG. 4 is a graph of data demonstrating the levels of Th2-type immuneresponses (as measured by production of IgG1 against an IMM antigen)stimulated by the antigen alone (AgE) and antigen conjugated to anon-stimulatory PN (mISS conj) in comparison to the vigorous Th1-typeimmune response stimulated in ISS-PN/IMM immunized mice. Antigen to PNratios are all 1:5. The horizontal axis represents the levels (units/ml)of antibody; the vertical axis shows the levels at 4 weeks followingprimary antigen exposure (shaded bars) and at 2 weeks followingsecondary antigen challenge (solid bars).

FIG. 5 is a graph of data demonstrating suppression of Th2 associatedanti-antigen (AgE) IgE production by ISS-PN/IMM in comparison to thelevels of IgE production stimulated by the antigen alone (AgE) and theantigen conjugated to a non-stimulatory PN (mISS conj). Antigen to PNratios are all 1:5. The horizontal axis represents the levels (countsper minute; cpm) of antibody; the vertical axis shows the levels at 4weeks following primary antigen exposure (shaded bars) and at 2 weeksfollowing secondary antigen challenge (solid bars).

FIG. 6 is a graph of data demonstrating the high levels of Th1associated interferon γ (IFNg) production stimulated by ISS-PN/IMM incomparison to the relatively low levels of the Th1 cytokine stimulatedby an ISS containing, antigen encoding plasmid (pACB-Z); the antigenalone (β-gal); the antigen mixed with an ISS; the antigen conjugated toa non-stimulatory PN (mISS conj); the antigen in adjuvant (alum) and,for reference, the IFNg levels in naive (unexposed) mice. Antigen to PNratios are all 1:5. The horizontal axis represents the levels (ng/ml) ofcytokine; the vertical axis shows the levels of cytokine at 4 weeksfollowing primary antigen exposure (shaded bars).

FIG. 7 is a graph of data demonstrating the vigorous antigen-specificcytotoxic T lymphocyte (CTL) response stimulated by ISS-PN/IMM incomparison to the levels of CTL production stimulated by an ISScontaining, antigen encoding plasmid (pACB-Z); the antigen alone(β-gal); the antigen mixed with an ISS; the antigen conjugated to anon-stimulatory PN (mISS conj); the antigen in adjuvant (alum) and, forreference, the CTL levels in naive (unexposed) mice. Antigen to PNratios are all 1:5. The horizontal axis represents the levels ofantigen-specific cell lysis obtained (as a percentage of control; noantigen); the vertical axis shows the levels of CTL detected atdifferent effector (antigen) to target ratios, from 0:1 to 10:1. Thelegend identifies how each cell population was treated.

DETAILED DESCRIPTION OF THE INVENTION

A. Biological Activity of the ISS-PN/IMM Conjugates

The immune response stimulated by the ISS-PN/IMM conjugates of theinvention differs from the vertebrate immune response to conventionalvaccination in both magnitude and quality. In the former respect, thehost immune response to an antigen is boosted to a level greater thanachieved on exposure to an ISS-PN or antigen administered alone ortogether in an unconjugated form. Thus, one surprising aspect of theinvention is that conjugation of an ISS-PN to an antigen-containing IMMproduces a synergism between the immunostimulatory activity of theISS-PN and the immunomodulatory activity of the IMM that immunizes thehost to the antigen more effectively than one would predict.

Advantageously, the immune response stimulated according to theinvention differs from the immune response of vertebrates toconventional vaccination in that the latter develops in a Th2 phenotypewhile the former develops in a Th1 phenotype. In this regard, it ishelpful to recall that CD4+ lymphocytes generally fall into one of twodistinct subsets; i.e., the Th1 and Th2 cells. Th1 cells principallysecrete IL-2, IFNγ and TNFβ (the latter two of which mediate macrophageactivation and delayed type hypersensitivity) while Th2 cellsprincipally secrete IL-4 (which stimulates production of IgEantibodies), IL-5 (which stimulates granulocyte infiltration of tissue),IL-6 and IL-10. These CD4+ subsets exert a negative influence on oneanother; i.e., secretion of Th1 lymphokines inhibits secretion of Th2lymphokines and vice versa.

Factors believed to favor Th1 activation resemble those induced by viralinfection and include intracellular pathogens, exposure to IFN-β, IFN-α,IFNγ, IL-12 and IL-18 and exposure to low doses of antigen. Th1 typeimmune responses also predominate in autoimmune disease. Factorsbelieved to favor Th2 activation include exposure to IL-4 and IL-10, APCactivity on the part of B lymphocytes and high doses of antigen.

Active Th1 (IFNγ) cells enhance cellular immunity and are therefore ofparticular value in responding to intracellular infections, while activeTh2 cells enhance antibody production and are therefore of value inresponding to extracellular infections (at the risk of anaphylacticevents associated with IL-4 stimulated induction of IgE antibodyproduction). Thus, the ability to shift host immune responses from theTh1 to the Th2 repertoire and vice versa has substantial clinicalsignificance for controlling host immunity against antigen challenge(e.g., in infectious and allergic conditions).

To that end, the methods of the invention shift the host immune responseto a sensitizing antigen toward a Th1 phenotype (Example I).Consequently, Th2 associated cytokine production and antigen stimulatedproduction of IgE (Examples II and III) are suppressed, thereby reducingthe host's risk of prolonged allergic inflammation and minimizing therisk of antigen-induced anaphylaxis. CTL production is also stimulatedto a greater degree in animals treated according to the invention.Because CTL production is tied to antigen processing in Class I MHCpathways, increased CTL production can be produced fromnon-Immunostimulatory PN/IMM as well as ISS-PN/IMM (Example IV).

Although the invention is not limited to any particular mechanism ofaction, it is conceivable that PN facilitate uptake of exogenous antigenby antigen presenting cells for presentation through host MHC Class Iprocessing pathways not normally stimulated by soluble antigen. Thus,ISS-PN/IMM carry antigen into MHC Class I processing pathways (which mayalso be achieved by PN/IMM without ISS activity) then stimulate acytokine cascade in a Th1 phenotype (as a result of ISS activity).Whatever the mechanism of action, use of ISS-PN/IMM to boost the host'simmune responsiveness to a sensitizing antigen and shift the immuneresponse toward a Th1 phenotype avoids the risk of immunization-inducedanaphylaxis, suppresses IgE production in response to a sensitizingantigen and eliminates the need to identify the sensitizing antigen foruse in immunization.

With reference to the invention, “boosting of immune responsiveness in aTh1 phenotype” in an ISS-PN/IMM treated host is evidenced by:

-   -   (1) a reduction in levels of IL-4 measured before and after        antigen-challenge; or detection of lower (or even absent) levels        of IL-4 in a treated host as compared to an antigen-primed, or        primed and challenged, control;    -   (2) an increase in levels of IL-12, IL-18 and/or IFN (α, β or γ)        before and after antigen challenge; or detection of higher        levels of IL-12, IL-18 and/or IFN (α, β or γ) in an ISS-PN/IMM        treated host as compared to an antigen-primed or, primed and        challenged, control;    -   (3) IgG2a antibody production in a treated host; or    -   (4) a reduction in levels of antigen-specific IgE as measured        before and after antigen challenge; or detection of lower (or        even absent) levels of antigen-specific IgE in an ISS-PN/IMM        treated host as compared to an antigen-primed, or primed and        challenged, control.

Exemplary methods for determining such values are described further inthe Examples.

Thus, the ISS-PN/IMM conjugates of the invention provide relativelysafe, effective means of stimulating a robust immune response in avertebrate host against any antigen.

B. ISS-PN/IMM Conjugates: Structure and Preparation

1. ISS-PN Root Structure

The ISS-ODN base of the ISS-PN/IMM conjugates of the invention includesan oligonucleotide, which may be a part of a larger nucleotide constructsuch as a plasmid. The term “polynucleotide” therefore includesoligonucleotides, modified oligonucleotides and oligonucleosides, aloneor as part of a larger construct. The polynucleotide may besingle-stranded DNA (ssDNA), double-stranded DNA (dsDNA),single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA).

The polynucleotide portion can be linearly or circularly configured, orthe oligonucleotide portion can contain both linear and circularsegments. Modifications of oligonucleotides include, but are not limitedto, modifications of the 3′OH or 5′OH group, modifications of thenucleotide base, modifications of the sugar component, and modificationsof the phosphate group.

The oligonucleotide base of ISS-PN/IMM conjugates may compriseribonucleotides (containing ribose as the only or principal sugarcomponent), deoxyribonucleotides deoxyribose as the principal sugarcomponent), or in accordance with established state-of-the-art modifiedsugars or sugar analogs may be incorporated in the oligonucleotide ofthe present invention. Thus, in addition to ribose and deoxyribose, thesugar moiety may be pentose, deoxypentose, hexose, deoxyhexos, glucose,arabinose, xylose, lyxose, and a sugar “analog” cyclopentyl group. Thesugar may be in pyranosyl or in a furanosyl form. In the modifiedoligonucleotides of the present invention the sugar moiety is preferablythe furanoside of ribose, deoxyribose, arabinose or 2′-0-methylribose,and the sugar may be attached to the respective heterocyclic baseseither in I or J anomeric configuration. The preparation of these sugarsor sugar analogs and the respective “nucleosides” wherein such sugars oranalogs are attached to a heterocyclic base (nucleic acid base) per seis known, and need not be described here, except to the extent suchpreparation may pertain to any specific example.

The phosphorous derivative (or modified phosphate group) which may beattached to the sugar or sugar analog moiety in the modifiedoligonucleotides of the present invention may be a monophosphate,diphosphate, triphosphate, alkylphosphate, alkanephosphate,phosphoronthioate, phosphorodithioate or the like. The preparation ofthe above-noted phosphate analogs, and their incorporation intonucleotides, modified nucleotides and oligonucleotides, per se, is alsoknown and need not be described here.

The heterocyclic bases, or nucleic acid bases which are incorporated inthe oligonucleotide base of the ISS-PN/IMM conjugates may be thenaturally occurring principal purine and pyrimidine bases, (namelyuracil or thymine, cytosine, adenine and guanine, as mentioned above),as well as naturally occurring and synthetic modifications of saidprincipal bases. Those skilled in the art will recognize that a largenumber of “synthetic” non-natural nucleosides comprising variousheterocyclic bases and various sugar moieties (and sugar analogs) havebecome available in the prior art, such that oligonucleotide base of theISS-PN/IMM conjugates may include one or several heterocyclic basesother than the principal five base components of naturally occurringnucleic acids. Preferably, however, the heterocyclic base in theoligonucleotide base of the ISS-PN/IMM conjugates is selected formuracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl,guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo[2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo [2.3-d] pyrimidin-3-ylgroups, where the purines are attached to the sugar moiety of theoligonucleotides via the 9-position, the pyrimidines via the 1-position,the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidinesvia the 1-position.

Structurally, the root oligonucleotide of the ISS-PN component ofISS-PN/IMM is a non-coding sequence which may include at least oneunmethylated CpG motif. The relative position of any CpG sequence inISS-PN with immunostimulatory activity in certain mammalian species(e.g., rodents) is 5′-CG-3′ (i.e., the C is in the 5′ position withrespect to the G in the 3′ position). PN/IMM can be convenientlyobtained by substituting the cytosine in the CpG dinucleotide withanother nucleotide; a particularly useful substitution is with a guanineto form GpG dinucleotide containing PN.

Some oligonucleotide ISS (ISS-ODN) are known. In such ISS-ODN, the CpGmotif is flanked by at least two purine nucleotides (e.g., GA or AA) andat least two pyrimidine nucleotides(5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′). CpGmotif-containing ISS-ODN are believed to stimulate B lymphocyteproliferation (see, e.g., Krieg, et al., Nature, 374:546–549, 1995).

The core hexamer structure of the foregoing ISS-PN may be flankedupstream and/or downstream by any number or composition of nucleotidesor nucleosides. However, ISS-PN are at least 6 bases in length, andpreferably are between 6 and 200 bases in length, to enhance uptake ofthe ISS-PN/IMM into target tissues. Those of ordinary skill in the artwill be familiar with, or can readily identify, reported nucleotidesequences of known ISS-ODN for reference in preparing ISS-PN. For easeof reference in this regard, the following sources are especiallyhelpful:

-   -   Yamamoto, et al., Microbiol. Immunol., 36:983 (1992)    -   Ballas, et al., J. Immunol., 157:1840 (1996)    -   Klinman, et al., J. Immunol., 158:3635 (1997)    -   Sato, et al., Science, 273:352 (1996)

Each of these articles are incorporated herein by reference for thepurpose of illustrating the level of knowledge in the art concerning thenucleotide composition of known ISS-ODN.

In particular, ISS-PN and PN useful in the invention include those whichhave the following hexameric nucleotide sequences:

-   -   1. For ISS-PN, hexamers having “CpG” motifs or, for PN, hexamers        having XpY motifs, where X cannot be C if Y is G and vice-versa;        and,    -   2. Inosine and/or uracil substitutions for nucleotides in the        foregoing hexamer sequences for use as RNA ISS-ODN.

For example, DNA based ISS-PN useful in the invention include thosewhich have the following hexameric nucleotide sequences:

(respectively, SEQ.ID.Nos. 1–18) AACGTT, AGCGTC, GACGTT, GGCGTT, AACGTC,AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT,GGCGCT, TTCGAA, GGCGTT and AACGCC.

RNA based ISS-PN useful in the invention include those which have thefollowing hexameric nucleotide sequences:

(respectively, SEQ.ID.Nos. 19–33) AACGUU, AACGpI, AACGpC, AGCGUC,AGCGpI, AGCGpC, GACGCU, GACGCpI, GACGCpC, GACGUU, GACGpI, GACGpC,GACGUC, GACGpI, GACGpC, and poly(I•C).

The ISS-PN may or may not include palindromic regions. If present, apalindrome may extend only to a CpG motif, if present, in the corehexamer sequence, or may encompass more of the hexamer sequence as wellas flanking nucleotide sequences.

In addition, backbone phosphate group modifications (e.g.,methylphosphonate, phosphorothioate, phosphoroamidate andphosphorodithioate internucleotide linkages) can confer anti-microbialactivity on the ISS-PN and enhance their stability in vivo, making themparticularly useful in therapeutic applications. A particularly usefulphosphate group modification is the conversion to the phosphorothioateor phosphorodithioate forms of ISS-PN. In addition to their potentiallyanti-microbial properties, phosphorothioates and phosphorodithioates aremore resistant to degradation in vivo than their unmodifiedoligonucleotide counterparts, making the ISS-PN/IMM of the inventionmore available to the host.

2. IMM Conjugate Partners.

The oligonucleotide base of the ISS-PN/IMM conjugate is conjugated to anIMM which includes an antigen and may further include animmunomodulatory agent. An “antigen” is a substance that is recognizedand bound specifically by an antibody or by a T cell antigen receptor.Antigens can include peptides, proteins, glycoproteins andpolysaccharides, including portions thereof and combinations thereof.The antigens can be those found in nature or can be synthetic.

The term “immunomodulatory” as used herein includes immunostimulatory aswell as immunosuppressive effects. Immunostimulatory effects include,but are not limited to, those that directly or indirectly enhancecellular or humoral immune responses. Examples of immunostimulatoryeffects include, but are not limited to, increased antigen-specificantibody production; activation or proliferation of a lymphocytepopulation such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes,macrophages and the like; as well as increased synthesis of Th1associated immunostimulatory cytokines including, but not limited to,IL-6, IL-12, IL-18, IFN-α, β and γ, TNF-α and the like.Immunosuppressive effects include those that directly or indirectlydecrease cellular or humoral immune responses.

Examples of immunosuppressive effects include, but are not limited to, areduction in antigen-specific antibody production such as reduced IgEproduction; activation of lymphocyte or other cell populations that haveimmunosuppressive activities such as those that result in immunetolerance; and increased synthesis of cytokines that have suppressiveeffects toward certain cellular functions. One example of this is IFN-γ,which can block IL-4 induced class switch to IgE and IgG1, therebyreducing the levels of these antibody subclasses.

Thus, an “immunomodulatory agent” suitable for use as conjugate partnersfor ISS-PN/IMM can be a peptide, such as an antigen or cytokine. Wherethe ISS-PN/IMM conjugate partner is a peptide, suitable peptides includepurified native peptides, synthetic peptides, recombinant proteins,crude protein extracts, attenuated or inactivated viruses, cells,micro-organisms, or fragments of such peptides.

Protein antigens that can serve as IMM conjugate partners includeantigens from a wide variety of sources, including allergens such asplant pollens, dust mite proteins, animal dander, saliva, and fungalspores as well as infectious microorganims. Examples of the latterinclude attenuated or inactivated viruses such as HIV-1, HIV-2,hepatitis, herpes simplex, rotavirus, polio virus, measles virus, humanand bovine papilloma virus, and slow brain viruses. For immunizationagainst tumor formation, the conjugate can include tumor cells (live orirradiated), tumor cell extracts, or protein subunits of tumor antigens.Vaccines for immuno-based contraception can be formed by including spermproteins as the peptide portion of the conjugate.

Among the suitable cytokines for use as components of IMM conjugatepartners are the interleukins (IL-1, IL-2, IL-3, etc.), interferons(e.g., IFN-α, IFN-β, IFN-γ), erythropoietin, colony stimulating factors(e.g., G-CSF, M-CSF, GM-CSF) and TNF-α.

IMM conjugate partners can also include amino acid sequences thatmediate protein binding to a specific receptor or that mediate targetingto a specific cell type or tissue. Examples include, but are not limitedto, antibodies or antibody fragments; peptide hormones such as humangrowth hormone; and enzymes. Co-stimulatory molecules such as B7 (CD80),trans-activating proteins such as transcription factors, chemokines suchas macrophage chemotactic protein (MCP) and other chemoattractant orchemotactic peptides are also useful peptide-based conjugate partners.

More specifically, suitable antigens for use as ISS-PN/IMM conjugatepartners include any molecule capable of being conjugated to anoligonucleotide and eliciting a B cell or T cell antigen-specificresponse. Preferably, antigens elicit an antibody response specific forthe antigen. A wide variety of molecules are antigens. These include,but are not limited to, sugars, lipids, autacoids and hormones, as wellas macromolecules such as complex carbohydrates, and phospholipids.Small molecules may need to be haptenized in order to be renderedantigenic.

Preferably the antigens are peptides, polysaccharides (such as thecapsular polysaccharides used in Haemophilus influenza vaccines),gangliosides and glycoproteins. The antigen may be an intact antigen orT cell epitope(s) of an antigen. These can be obtained through severalmethods known in the art, including isolation and synthesis usingchemical and enzymatic methods. In certain cases, such as for manysterols fatty acids and phospholipids, the antigenic portions arecommercially available.

Many antigenic peptides and proteins are known in, and available to theart; others can be identified using conventional techniques. Examples ofknown antigens include, but are not limited to:

-   -   a. Allergens such as reactive major dust mite allergens Der pI        and Der pII (see, Chua, et al., J. Exp. Med., 167:175–182, 1988;        and, Chua, et al., Int. Arch. Allergy Appl. Immunol.,        91:124–129, 1990), T cell epitope peptides of the Der pII        allergen (see, Joost van Neerven, et al., J. Immunol.,        151:2326–2335, 1993), the highly abundant Antigen E (Amb aI)        ragweed pollen allergen (see, Rafnar, et al., J. Biol. Chem.,        266:1229–1236, 1991), phospholipase A₂ (bee venom) allergen and        T cell epitopes therein (see, Dhillon, et al., J. Allergy Clin.        Immunol., ___:42–___, 1992), white birch pollen (Betvl) (see,        Breiteneder, et al., EMBO, 8:1935–1938, 1989), the Fel dI major        domestic cat allergen (see, Rogers, et al., Mol. Immunol.,        30:559–568, 1993), tree pollen (see, Elsayed et al., Scand. J.        Clin. Lab. Invest. Suppl., 204:17–31, 1991) and grass pollen        (see, Malley, J. Reprod. Immunol., 16:173–86, 1989).    -   b. Live, attenuated and inactivated microorganisms such as        inactivated polio virus (Jiang et al., J. Biol. Stand.,        14:103–9, 1986), attenuated strains of Hepatitis A virus        (Bradley et al., J. Med. Virol., 14:373–86, 1984), attenuated        measles virus (James et al., N. Engl. J. Med., 332:1262–6, 1995)        and epitopes of pertussis virus (e.g., ACEL-IMUNE® acellular        DTP, Wyeth-Lederle Vaccines and Pediatrics).    -   C. Contraceptive antigens such as human sperm protein (Lea et        al., Biochim. Biophys. Acta, 1307:263, 1996).

The published sequence data and methods for isolation and synthesis ofthe antigens described in these articles are incorporated herein by thisreference to illustrate knowledge in the art regarding useful antigensources. Those of ordinary skill in the art will be familiar with, orcan readily ascertain, the identity of other useful antigens for use asISS-PN/IMM conjugate partners.

Particularly useful immunostimulatory peptides for inclusion in IMM arethose which stimulate Th1 immune responses, such as IL-12 (Bliss, etal., J. Immunol., 156:887–894, 1996), IL-18, INF-α,β and γ or TGF-α.Conjugation of adjuvants (such as keyhole limpet hemocyanin, KLH) to theISS-PN/IMM conjugate can further enhance the activity of the ISS-PN/IMMconjugates of the invention.

Other useful adjuvants include cholera toxin, procholeragenoid, choleratoxin B subunit and fungal polysaccharides including, but not limitedto, schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives,phorbol esters, microspheres, non-Helicobacter pylori bacterial lysates,labile toxin of Escherichia coli, block polymers, saponins, and ISCOMs.For additional adjuvants, those of ordinary skill in the art may alsorefer to, for example, Azuma, I., “Synthetic Immunoadjuvants:Application to Non-Specific Host Stimulation and Potentiation of VaccineImmunogenicity” Vaccine, vol. 10, 1000 (1992); Pockley, A. G. &Montgomery, P. C., “In vivo Adjuvant Effect of Interleukins 5 and 6 onRat Tear IgA Antibody Responses” Immunology, vol. 73, 19–23 (1991);Adam, A. & Lederer, E. “Muramyl peptides as Immunomodulators” ISI ATLASOF SCIENCE 205 (1988); Clements, J. D., et al. “Adjuvant Activity ofEscherichia coli Heat-labile Enterotoxin and Effect on the Induction ofOral Tolerance in Mice to Unrelated Protein Antigens” Vaccine, vol. 6,269 (1988); Ben Ahmeida, E. T. S., et al. “Immunopotentiation of Localand Systemic Humoral Immune Responses by ISCOMs, Liposomes and FCA: Rolein Protection Against Influenza A in Mice” Vaccine, vol. 11, 1302(1993); and Gupta, R. K. et al. “Adjuvants—A Balance Between Toxicityand Adjuvanticity” Vaccine, vol. 11, 290–308 (1993).

Those of ordinary skill in the art will appreciate that non-antigencomponents of IMM described above can also be administered inunconjugated form with an ISS-PN/IMM (antigen only) conjugate. Thus, theco-administration of such components is encompassed by the invention.

C. Synthesis of Polynucleotide Conjugates

1. Polynucleotide Portion

ISS-PN can be synthesized using techniques and nucleic acid synthesisequipment which are well-known in the art. For reference in this regard,see e.g., Ausubel, et al., Current Protocols in Molecular Biology, Chs.2 and 4 (Wiley Interscience, 1989); Maniatis, et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., New York, 1982); U.S. Pat.No. 4,458,066 and U.S. Pat. No. 4,650,675. When assembled enzymatically,the individual units can be ligated with a ligase such as T4 DNA or RNAligase as described in, for example, U.S. Pat. No. 5,124,246.Oligonucleotide degradation could be accomplished through the exposureof an oligonucleotide to a nuclease, as exemplified in U.S. Pat. No.4,650,675. These references are incorporated herein by reference for thesole purpose of demonstrating knowledge in the art concerning productionof synthetic polynucleotides. Because the ISS-PN is non-coding, there isno concern about maintaining an open reading frame during synthesis.

Alternatively, ISS-PN may be isolated from microbial species (especiallymycobacteria) using techniques well-known in the art, such as nucleicacid hybridization. Preferably, such isolated ISS-PN will be purified toa substantially pure state; i.e., to be free of endogenous contaminants,such, as lipopolysaccharides. ISS-PN isolated as part of a largerpolynucleotide can be reduced to the desired length by techniques wellknown in the art, such as by endonuclease digestion. Those of ordinaryskill in the art will be familiar with, or can readily ascertain,techniques suitable for isolation, purification and digestion ofpolynucleotides to obtain ISS-PN of potential use in the invention.

Circular ISS-PN can be isolated, synthesized through recombinantmethods, or chemically synthesized. Where the circular ISS-PN isobtained through isolation or through recombinant methods, the ISS-PNwill preferably be a plasmid. The chemical synthesis of smaller circularoligonucleotides can be performed using literature methods (Gao et al.,Nucleic Acids Res. (1995) 23:2025–9; Wang et al., Nucleic Acids Res.(1994) 22:2326–33).

The ISS-PN can also contain modified oligonucleotides. These modifiedoligonucleotides can be synthesized using standard chemicaltransformations. The efficient solid-support based construction ofmethylphosphonates has been described. Agrawal et al. (19) Tet. Lett.28:3539–3542. The synthesis of other phosphorous based modifiedoligonucleotides, such as phosphotriesters (Miller et al. JACS 93,6657–6665), phosphoramidates (Jager et al, Biochemistry 27, 7247–7246),and phosphorodithioates (U.S. Pat. No. 5,453,496) has also beendescribed. Other non-phosphorous based modified oligonucleotides canalso be used (Stirchak et al., Nucleic Acids Res. 17, 6129–6141).

The preparation of base-modified nucleosides, and the synthesis ofmodified oligonucleotides using said base-modified nucleosides asprecursors, has been described, for example, in U.S. Pat. Nos.4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosideshave been designed so that they can be incorporated by chemicalsynthesis into either terminal or internal positions of anoligonucleotide. Such base-modified nucleosides, present at eitherterminal or internal positions of an oligonucleotide, can serve as sitesfor attachment of a peptide or other antigen. Nucleosides modified intheir sugar moiety have also bee described (e.g., U.S. Pat. Nos.4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be used similarly.

The techniques for making phosphate group modifications tooligonucleotides are known in the art and do not require detailedexplanation. For review of one such useful technique, the anintermediate phosphate triester for the target oligonucleotide productis prepared and oxidized to the naturally occurring phosphate triesterwith aqueous iodine or with other agents, such as anhydrous amines. Theresulting oligonucleotide phosphoramidates can be treated with sulfur toyield phophorothioates. The same general technique (excepting the sulfurtreatment step) can be applied to yield methylphosphoamidites frommethylphosphonates. For more details concerning phosphate groupmodification techniques, those of ordinary skill in the art may wish toconsult U.S. Pat. Nos. 4,425,732; 4,458,066; 5,218,103 and 5,453,496, aswell as Tetrahedron Lett. at 21:4149 (1995), 7:5575 (1986), 25:1437(1984) and Journal Am. ChemSoc., 93:6657 (1987), the disclosures ofwhich are incorporated herein for the sole purpose of illustrating thestandard level of knowledge in the art concerning preparation of thesecompounds.

2. Linking the PN Component to the IMM Component

The ISS-PN component can be linked to the IMM portion of the conjugatein a variety of ways. The link can be made at the 3′ or 5′ end of theISS-PN, or to a suitably modified base at an internal position in thePN. If the peptide contains a suitable reactive group (e.g., anN-hydroxysuccinimide ester) it can be reacted directly with the N⁴ aminogroup of cytosine residues. Depending on the number and location ofcytosine residues in the ISS-PN, specific labeling at one or moreresidues can be achieved.

Alternatively, modified oligonucleosides, such as are known in the art,can be incorporated at either terminus, or at internal positions in theISS-PN. These can contain blocked functional groups which, whendeblocked, are reactive with a variety of functional groups which can bepresent on, or attached to, a peptide of interest.

The IMM portion of the conjugate can be attached to the 3′-end of theISS-PN through solid support chemistry. For example, the ISS-PN portioncan be added to a polypeptide portion that has been pre-synthesized on asupport (Haralambidis et al., Nucleic Acids Res. (1990) 18:493–99;Haralambidis et al., Nucleic Acids Res. (1990) 18:501–505).Alternatively, the PN can be synthesized such that it is connected to asolid support through a cleavable linker extending from the 3′-end. Uponchemical cleavage of the ISS-PN from the support, a terminal thiol groupis left at the 3′-end of the ISS-PN (Zuckermann et al., Nucleic AcidsRes. (1987) 15:5305–5321; Corey et al., (1987) Science 238:1401–1403),or a terminal amine group is left at the 3′-end of the PN (Nelson etal., Nucleic Acids Res. (1989) 17:1781–94). Conjugation of theamino-modified PN to amino groups of the peptide can be performed asdescribed in Benoit et al., Neuromethods (1987) 6:43–72. Conjugation ofthe thiol-modified ISS-PN to carboxyl groups of the peptide can beperformed as described in Sinah et al., Oligonucleotide Analogues: APractical Approach (1991) IRL Press.

The IMM portion of the conjugate can be attached to the 5′-end of theISS-PN through an amine, thiol, or carboxyl group that has beenincorporated into the ISS-PN during its synthesis. Preferably, while theISS-PN is fixed to the solid support, a linking group comprising aprotected amine, thiol, or carboxyl at one end, and a phosphoramidite atthe other, is covalently attached to the 5′-hydroxyl (Agrawal et al.,Nucleic Acids Res. (1986) 14:6227–6245; Connolly, Nucleic Acids Res.(1985) 13:4485–4502; Coull et al., Tetrahedron Lett. (1986)27:3991–3994; Kremsky et al., Nucleic Acids Res. (1987) 15:2891–2909;Connolly, Nucleic Acids Res. (1987) 15:3131–3139; Bischoff et al., Anal.Biochem. (1987) 164:336–344; Blanks et al., Nucleic Acids Res. (1988)16:10283–10299; U.S. Pat. Nos. 4,849,513, 5,015,733, 5,118,800, and5,118,802). Subsequent to deprotection, the latent amine, thiol, andcarboxyl functionalities can be used to covalently attach the PN to apeptide (Benoit et al., Neuromethods (1987) 6:43–72; Sinah et al.,Oligonucleotide Analogues: A Practical Approach (1991) IRL Press).

A peptide portion can be attached to a modified cytosine or uracil atany position in the ISS-PN. The incorporation of a “linker arm,”possessing a latent reactive functionality, such as an amine or carboxylgroup, at C-5 of the modified base provides a handle for the peptidelinkage (Ruth, 4th Annual Congress for Recombinant DNA Research, p.123).

The linkage of the ISS-PN to a peptide can also be formed through ahigh-affinity, non-covalent interaction such as a biotin-streptavidincomplex. A biotinyl group can be attached, for example, to a modifiedbase of an oligonucleotide (Roget et al., Nucleic Acids Res. (1989)17:7643–7651). Incorporation of a streptavidin moiety into the peptideportion allows formation of a non-covalently bound complex of thestreptavidin conjugated peptide and the biotinylated PN.

The linkage of the ISS-PN to a lipid can be formed using standardmethods. These methods include, but are not limited to, the synthesis ofoligonucleotide-phospholipid conjugates (Yanagawa et al., Nucleic AcidsSymp. Ser. (1988) 19:189–92), oligonucleotide-fatty acid conjugates(Grabarek et al., Anal. Biochem. (1990) 185:131–35; Staros et al., Anal.Biochem. (1986) 156:220–22), and oligonucleotide-sterol conjugates(Boujrad et al., Proc. Natl. Acad. Sci. USA (1993) 90:5728–31).

The linkage of the ISS-PN to a oligosaccharide can be formed usingstandard known methods. These methods include, but are not limited to,the synthesis of oligonucleotide-oligosaccharide conjugates wherein theoligosaccharide is a moiety of an immunoglobulin (O'Shannessy et al., J.Applied Biochem. (1985) 7:347–55).

Adjuvants and cytokies may also be genetically or chemically linked tothe ISS-ODN conjugates. Examples of this type of fusion peptide areknown to those skilled in the art and can also be found in Czerkinsky etal., Infect. Immun., 57: 1072–77 (1989); Nashar et al., Vaccine, 11:235–40 (1993); and Dertzbaugh and Elson, Infect. Immun., 61: 48–55(1993).

The linkage of a circular ISS-PN to an IMM can be formed in severalways. Where the circular PN is synthesized using recombinant or chemicalmethods, a modified nucleoside (Ruth, in Oligonucleotides and Analogues:A Practical Approach (1991) IRL Press). Standard linking technology canthen be used to connect the circular ISS-PN to the antigen orimmunostimulatory peptide (Goodchild, Bioconjugate Chem. (1990) 1: 165).Where the circular ISS-PN is isolated, or synthesized using recombinantor chemical methods, the linkage can be formed by chemically activating,or photoactivating, a reactive group (e.g. carbene, radical) that hasbeen incorporated into the antigen or immunostimulatory peptide.

Additional methods for the attachment of peptides and other molecules toISS-PNs can be found in C. Kessler: Nonradioactive labeling methods fornucleic acids in L. J. Kricka (ed.) “Nonisotopic DNA Probe Techniques,”Academic Press 1992 and in Geoghegan and Stroh, Bioconjug. Chem.,3:138–146, 1992.

D. Methods and Routes for Administration of ISS-PN/IMM to a Host

1. Drug Delivery

The ISS-PN/IMM of the invention are administered to a host using anyavailable method and route suitable for drug delivery, including ex vivomethods (e.g., delivery of cells incubated or transfected with anISS-PN/IMM) as well as systemic or localized routes. However, those ofordinary skill in the art will appreciate that methods and localizedroutes which direct the ISS-PN/IMM into antigen-sensitized tissue willbe preferred in most circumstances to systemic routes of administration,both for immediacy of therapeutic effect and avoidance of in vivodegradation.

The entrance point for many exogenous antigens into a host is throughthe skin or mucosa. Thus, delivery methods and routes which target theskin (e.g., for cutaneous and subcutaneous conditions) or mucosa (e.g.,for respiratory, ocular, lingual or genital conditions) will beespecially useful. Those of ordinary skill in the clinical arts will befamiliar with, or can readily ascertain, means for drug delivery intoskin and mucosa. For review, however, exemplary methods and routes ofdrug delivery useful in the invention are briefly discussed below.

Intranasal administration means are particularly useful in addressingrespiratory inflammation, particularly inflammation mediated by antigenstransmitted from the nasal passages into the trachea or broncheoli. Suchmeans include inhalation of aerosol suspensions or insufflation of thepolynucleotide compositions of the invention. Nebulizer devices suitablefor delivery of polynucleotide compositions to the nasal mucosa, tracheaand bronchioli are well-known in the art and will therefore not bedescribed in detail here. For general review in regard to intranasaldrug delivery, those of ordinary skill in the art may wish to consultChien, Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992).

Dermal routes of administration, as well as subcutaneous injections, areuseful in addressing allergic reactions and inflammation in the skin.Examples of means for delivering drugs to the skin are topicalapplication of a suitable pharmaceutical preparation, transdennaltransmission, injection and epidermal administration.

For transdermal transmission, absorption promoters or iontophoresis aresuitable methods. For review regarding such methods, those of ordinaryskill in the art may wish to consult Chien, supra at Ch. 7.Iontophoretic transmission may be accomplished using commerciallyavailable “patches” which deliver their product continuously viaelectric pulses through unbroken skin for periods of several days ormore. Use of this method allows for controlled transmission ofpharmaceutical compositions in relatively great concentrations, permitsinfusion of combination drugs and allows for contemporaneous use of anabsorption promoter.

An exemplary patch product for use in this method is the LECTRO PATCHtrademarked product of General Medical Company of Los Angeles, Calif.This product electronically maintains reservoir electrodes at neutral pHand can be adapted to provide dosages of differing concentrations, todose continuously and/or to dose periodically. Preparation and use ofthe patch should be performed according to the manufacturer's printedinstructions which accompany the LECTRO PATCH product; thoseinstructions are incorporated herein by this reference.

Epidermal administration essentially involves mechanically or chemicallyirritating the outermost layer of the epidermis sufficiently to provokean immune response to the irritant. An exemplary device for use inepidermal administration employs a multiplicity of very narrow diameter,short tynes which can be used to scratch ISS-PN/IMM coated onto thetynes into the skin. The device included in the MONO-VACC old tuberculintest manufactured by Pasteur Merieux of Lyon, France is suitable for usein epidermal administration of ISS-PN/IMM. Use of the device isaccording to the manufacturer's written instructions included with thedevice product; these instructions regarding use and administration areincorporated herein by this reference to illustrate conventional use ofthe device. Similar devices which may also be used in this embodimentare those which are currently used to perform allergy tests.

Opthalmic administration (e.g., for treatment of allergicconjunctivitis) involves invasive or topical application of apharmaceutical preparation to the eye. Eye drops, topical cremes andinjectable liquids are all examples of suitable mileaus for deliveringdrugs to the eye.

Systemic administration involves invasive or systemically absorbedtopical administration of pharamaceutical preparations. Topicalapplications as well as intravenous and intramuscular injections areexamples of common means for systemic administration of drugs.

2. Dosing Parameters

A particular advantage of the ISS-PN/IMM of the invention is theircapacity to exert immunomodulatory activity even at relatively minutedosages. Although the dosage used will vary depending on the clinicalgoals to be achieved, a suitable dosage range is one which provides upto about 1–1000 μg of ISS-PN/IMM/ml of carrier in a single dosage.Alternatively, a target dosage of ISS-PN/IMM can be considered to beabout 1–10 μM in a sample of host blood drawn within the first 24–48hours after administration of ISS-PN/IMM. Based on current studies,ISS-PN/IMM are believed to have little or no toxicity at these dosagelevels.

In this respect, it should be noted that the anti-inflammatory andimmunotherapeutic activity of ISS-PN/IMM in the invention is essentiallydose-dependent. Therefore, to increase ISS-PN/IMM potency by a magnitudeof two, each single dose is doubled in concentration. Clinically, it maybe advisable to administer the ISS-PN/IMM in a low dosage (e.g., about 1μg/ml to about 50 μg/ml), then increase the dosage as needed to achievethe desired therapeutic goal.

In view of the teaching provided by this disclosure, those of ordinaryskill in the clinical arts will be familiar with, or can readilyascertain, suitable parameters for administration of ISS-PN/IMMaccording to the invention.

3. ISS-PN/IMM Compositions

ISS-PN/IMM will be prepared in a pharmaceutically acceptable compositionfor delivery to a host. Pharmaceutically acceptable carriers preferredfor use with the ISS-PN/IMM of the invention may include sterile aqueousof non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. A composition of ISS-PN/IMM may also be lyophilizedusing means well known in the art, for subsequent reconstitution and useaccording to the invention.

Absorption promoters, detergents and chemical irritants (e.g.,keritinolytic agents) can enhance transmission of an ISS-PN/IMMcomposition into a target tissue. For reference concerning generalprinciples regarding absorption promoters and detergents which have beenused with success in mucosal delivery of organic and peptide-baseddrugs, see Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker,1992).

Examples of suitable nasal absorption promoters in particular are setforth at Chien, supra at Ch. 5, Tables 2 and 3; milder agents arepreferred. Suitable agents for use in the method of this invention formucosal/nasal delivery are also described in Chang, et al., Nasal DrugDelivery, “Treatise on Controlled Drug Delivery”, Ch. 9 and Table 3–4Bthereof, (Marcel Dekker, 1992). Suitable agents which are known toenhance absorption of drugs through skin are described in Sloan, Use ofSolubility Parameters from Regular Solution Theory to DescribePartitioning-Driven Processes, Ch. 5, “Prodrugs: Topical and Ocular DrugDelivery” (Marcel Dekker, 1992), and at places elsewhere in the text.All of these references are incorporated herein for the sole purpose ofillustrating the level of knowledge and skill in the art concerning drugdelivery techniques.

A colloidal dispersion system may be used for targeted delivery of theISS-PN/IMM to specific tissue. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome.

Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2–4.0 μm can encapsulate asubstantial percentage of an aqueous buffer containing largemacromolecules. RNA, DNA and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition tomammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the antisense polynucleotides at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14–18carbon atoms, particularly from 16–18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various well known linking groups can be used forjoining the lipid chains to the targeting ligand (see, e.g., Yanagawa,et al., Nuc. Acids Symp. Ser., 19:189 (1988); Grabarek, et al., Anal.Biochem., 185:131 (1990); Staros, et al., Anal. Biochem., 156:220 (1986)and Boujrad, et al., Proc. Natl. Acad. Sci. USA, 90:5728 (1993), thedisclosures of which are incorporated herein by reference solely toillustrate the standard level of knowledge in the art concerningconjugation of PNs to lipids). Targeted delivery of ISS-PN/IMM can alsobe achieved by conjugation of the ISS-PN/IMM to a the surface of viraland non-viral recombinant expression vectors, to an antigen or otherligand, to a monoclonal antibody or to any molecule which has thedesired binding specificity.

Co-administration of a peptide drug with an ISS-PN/IMM according to theinvention may also be achieved by incorporating the ISS-PN/IMM in cis orin trans into a recombinant expression vector (plasmid, cosmid, virus orretrovirus) which codes for any therapeutically beneficial proteindeliverable by a recombinant expression vector. If incorporation of anISS-PN/IMM into an expression vector for use in practicing the inventionis desired, such incorporation may be accomplished using conventionaltechniques which do not require detailed explanation to one of ordinaryskill in the art. For review, however, those of ordinary skill may wishto consult Ausubet, Current Protocols in Molecular Biology, supra.

D. Screening for Active ISS-PN/IMM

Confirmation that a particular compound has the properties of anISS-PN/IMM useful in the invention can be obtained by evaluating whetherthe ISS-PN/IMM affects cytokine secretion and IgG antibody isotypeproduction as described in Section A.I, above. Details of in vitrotechniques useful in making such an evaluation are given in theExamples; those of ordinary skill in the art will also know of, or canreadily ascertain, other methods for measuring cytokine secretion andantibody production along the parameters taught herein.

E. Kits for Use in Practicing the Methods of the Invention

For use in the methods described above, kits are also provided by theinvention. Such kits may include any or all of the following: ISS-PN/IMM(conjugated or unconjugated); a pharmaceutically acceptable carrier (maybe pre-mixed with the ISS-PN/IMM) or suspension base for reconstitutinglyophilized ISS-PN/IMM; additional medicaments; a sterile vial for eachISS-PN/IMM and additional medicament, or a single vial for mixturesthereof; device(s) for use in delivering ISS-PN/IMM to a host; assayreagents for detecting indicia that the anti-inflammatory and/orimmunostimulatory effects sought have been achieved in treated animalsand a suitable assay device.

Examples illustrating the practice of the invention are set forth below.The examples are for purposes of reference only and should not beconstrued to limit the invention, which is defined by the appendedclaims. All abbreviations and terms used in the examples have theirexpected and ordinary meaning unless otherwise specified.

EXAMPLE I Selective Ineuction of a Th1 Response in a Host AfterAdministration of an ISS-PN/IMM

In mice, IgG 2A antibodies are serological markers for a Th1 type immuneresponse, whereas IgG 1 antibodies are indicative of a Th2 type immuneresponse. Th2 responses include the allergy-associated IgE antibodyclass; soluble protein antigens tend to stimulate relatively strong Th2responses. In contrast, Th1 responses are induced by antigen binding tomacrophages and dendritic cells.

To determine which response, if any, would be produced by mice whoreceived ISS-PN/IMM according to the invention, eight groups of Balb/cmice were immunized with 10 μg β-galactosidase protein (conjugated toavidin; Sigma, St. Louis, Mo.) to produce a model allergic phenotype. Asset forth in the Table below, some of the mice received antigen alone,some received an antigen-ISS-PN conjugate or a conjugate using a mutant,non-stimulatory PN as a conjugate for the antigen, and others receivedthe antigen in an unconjugated mixture with an ISS-PN. Naive mice areshown for reference:

Mouse Group ISS-PN/IMM Treatment 1 None (β-gal antigen vaccinated) 2DY1018-βgal conjugate (ISS-PN/IMM) 3 DY1019-βgal conjugate (PN/IMM) 4DY1018 mixed with βgal (unconjugated) 5 βgal in adjuvant (alum) 6plasmid DNA (ISS-ODN present but not expressible with antigen) 7 naivemice (no antigen priming)DY1018 has the nucleotide sequence:

5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO:1) with a phosphothioatebackbone and DY 1019 has the nucleotide sequence:

5′-TGACTGTGAAGGTTGGAGATGA-3′ (SEQ ID NO:2) with a phosphothioatebackbone.

At 2 week intervals, any IgG 2a and IgG 1 to β-galactosidase present inthe serum of each mouse were measured by enzyme-linked immunoabsorbentassay (using antibodies specific for the IgG 1 and IgG 2A subclasses) onmicrotiter plates coated with the enzyme.

As shown in FIG. 1, only the mice who received the ISS-PN/IMM producedhigh titers of IgG 2A antibodies, which increased in number over aperiod of 8 weeks. As shown in FIG. 2, immunization of the mice with theantigen itself or with the PN/IMM induced production of relatively hightiters of IgG 1 antibodies. The data shown in the FIGURES compriseaverages of the values obtained from each group of mice.

To evaluate the effect of treatment of a host before and after asecondary antigen challenge, 3 groups of Balb/c mice were immunized with10 μg of antigen E (AgE) in alum to produce a model allergic phenotypeand challenged again with the antigen, ISS-PN/IMM or mutant(nonstimulatory) PN/IMM at 5 weeks post-priming. An ELISA for IgG1 andIgG2a antibodies was performed as described 4 weeks after priming (oneweek before secondary antigen challenge) and again at 7 weeks (2 weeksafter secondary challenge).

Again, the mice who received the ISS-PN/IMM mounted a strong Th1 typeresponse to the antigen (IMM) as compared to the antigen-immunized andmutant PN/IMM immunized mice (FIG. 3), while the reverse was true of aTh2 type response in the same mice (FIG. 4).

These data indicate that a selective Th1 response is induced byadministration of an ISS-PN/IMM according to the invention to both anantigen-primed (pre-antigen challenge) and an antigen-challenged host.

EXAMPLE II Suppression of IgE Antibody Response to Antigen byImmunization with ISS-PN/IMM

To demonstrate the IgE suppression achieved through stimulation of a Th1type cellular immune response in preference to a Th2 type cellularimmune response, five to eight week old Balb/c mice were immunized withAgE as described in the previous Example.

IgE anti-Age were detected using a solid phase radioimmunoassay (RAST)in a 96 well polyvinyl plate (a radioisotopic modification of the ELISAprocedure described in Coligan, “Current Protocols In Immunology”, Unit7.12.4, Vol. 1, Wiley & Sons, 1994), except that purified polyclonalgoat antibodies specific for mouse ε chains were used in lieu ofantibodies specific for human Fab. To detect anti-AgE IgE, the plateswere coated with AgE (10 μg/ml). The lowest IgE concentration measurableby the assay employed was 0.4 ng of IgE/ml.

Measuring specifically the anti-antigen response by each group of mice,as shown in FIG. 5, anti-AgE IgE levels in the ISS-PN/IMM immunized micewere consistently low both before and after boosting, while the proteinand mutant ISS-PN/IMM injected mice developed high levels of anti-AgEafter antigen challenge.

These data show that the ISS-PN/IMM immunized mice developed an antigenspecific Th1 response (suppressing the Th2 IgE response) to the antigen.

EXAMPLE III INFγ Levels in Mice after Delivery of ISS-PN/IMM

BALB/c mice were immunized with βgal as described in Example I thensacrificed 24 hrs later. Splenocytes were harvested from each mouse.

96 well microtiter plates were coated with anti-CD3 antibody(Pharmingen, La Jolla, Calif.) at a concentration of 1 μg/ml of saline.The anti-CD3 antibody stimulates T cells by delivering a chemical signalwhich mimicks the effects of binding to the T cell receptor (TCR)complex. The plates were washed and splenocytes added to each well(4×105/well) in a medium of RPMI 1640 with 10% fetal calf serum.Supernatants were obtained at days 1, 2 and 3.

Th1 cytokine (INFγ) levels were assayed with an anti-INFγ murineantibody assay (see, e.g., Coligan, “Current Protocols in Immunology”,Unit 6.9.5., Vol. 1, Wiley & Sons, 1994). Relatively low levels of INF-γwould be expected in mice with a Th2 phenotype, while relatively highlevels of INF-γ would be expected in mice with a Th1 phenotype.

As shown in FIG. 5, levels of Th1 stimulated IFN-γ secretion weregreatly increased in the ISS-PN/IMM treated mice, but substantiallyreduced in each other set of mice (as compared to the control),indicating development of a Th2-type phenotype in the latter mice and aTh1 phenotype in the ISS-PN/IMM treated mice.

EXAMPLE IV Boosting of CTL Responses by ISS-PN/IMM

A mixture of lymphoytes was obtained and contacted with βgal antigenalone or as part of the constructs and mixtures described in Example I.As shown in FIG. 6, CTL production in response to ISS-PN/IMM wasconsistently higher than the response to antigen delivered in otherforms; even twice as high than in animals treated with an unconjugatedmixture of ISS-PN and IMM antigen.

In the experiment, the higher values for the mice treated withM-ISS-PN/IMM after antigen challenge as compared to the conventionallyimmunized mice is most likely owing to the antigen carrier properties ofDY1019.

Thus, longer-term immunity mediated by cellular immune responses isbenefitted by treatment according to the invention.

1. A method of reducing IgE production in a mammal comprisingadministering an immunomodulatory composition to the mammal in an amountsufficient to reduce IgE production in the mammal, wherein theimmunomodulatory composition comprises an immunomodulatory molecule,wherein said immunomodulatory molecule comprises an antigen and isconjugated to an immunostimulatory polynucleotide (ISS-PN), said ISS-PNcomprising an immunostimulatory sequence (ISS) comprising the sequence5′-cytosine, guanine-3′.
 2. The method of claim 1, wherein the ISS is atleast six nucleotides in length.
 3. The method of claim 1, wherein theISS comprises the sequence 5′-Purine, Purine, C, G, Pyrimidine,Pyrimidine-3′.
 4. The method of claim 1, wherein the polynucleotidefurther comprises a phosphate backbone modification.
 5. The method ofclaim 4, wherein the phosphate backbone modification is phosphorothioateor phosphorodithioate.
 6. The method of claim 1, wherein said ISS-PN isfrom 6 to about 200 nucleotides in length.
 7. The method of claim 1,wherein the ISS comprises a sequence selected from the group consistingof AACGTT, AGCGTT, and GACGTT.
 8. The method of claim 1, wherein the ISScomprises the sequence TGACTGTGAACGTTCGAGATGA (SEQ ID NO:1).
 9. Themethod of claim 1, wherein the composition further comprises anadjuvant.
 10. The method of claim 1, wherein the antigen is covalentlyconjugated to the polynucleotide.
 11. The method of claim 1, wherein theantigen is non-covalently conjugated to the polynucleotide.
 12. Themethod of claim 1, wherein the antigen is conjugated by a linker arm tothe polynucleotide.
 13. A method for reducing antigen-stimulatedinflammation in a mammal comprising administering an immunomodulatorycomposition to the mammal in an amount sufficient to reduce inflammationstimulated by the antigen in the mammal, wherein the immunomodulatorycomposition comprises an immunomodulatory molecule, wherein saidimmunomodulatory molecule comprises an antigen and is conjugated to animmunostimulatory polynucleotide (ISS-PN), said ISS-PN comprising animmunostimulatory sequence (ISS) comprising the sequence 5′-cytosine,guanine-3′.
 14. The method of claim 13, wherein the ISS is at least sixnucleotides in length.
 15. The method of claim 13, wherein the ISScomprises the sequence 5′-Purine, Purine, C, G, Pyrimidine,Pyrimidine-3′.
 16. The method of claim 13, wherein the polynucleotidefurther comprises a phosphate backbone modification.
 17. The method ofclaim 13, wherein the composition further comprises an adjuvant.
 18. Themethod of claim 13, wherein the antigen is covalently conjugated to thepolynucleotide.
 19. The method of claim 13, wherein the antigen isnon-covalently conjugated to the polynucleotide.
 20. The method of claim13, wherein the antigen is conjugated by a linker arm to thepolynucleotide.
 21. A method of suppressing a Th2 immune response in amammal comprising an immunomodulatory composition in an amountsufficient to suppress the Th2 immune response, wherein theimmunomodulatory composition comprises an immunomodulatory molecule,wherein said immunomodulatory molecule comprises an antigen and isconjugated to an immunostimulatory polynucleotide (ISS-PN), said ISS-PNcomprising an immunostimulatory sequence (ISS) comprising the sequence5′-cytosine, guanine-3′.
 22. The method of claim 21, wherein the ISS isat least six nucleotides in length.
 23. The method of claim 21, whereinthe ISS comprises the sequence 5′-Purine, Purine, C, G, Pyrimidine,Pyrimidine-3′.
 24. The method of claim 21, wherein the polynucleotidefurther comprises a phosphate backbone modification.
 25. The method ofclaim 21, wherein the composition further comprises an adjuvant.
 26. Themethod of claim 21, wherein the antigen is covalently conjugated to thepolynucleotide.
 27. The method of claim 21, wherein the antigen isnon-covalently conjugated to the polynucleotide.
 28. The method of claim21, wherein the antigen is conjugated by a linker arm to thepolynucleotide.
 29. A method of stimulating a Th1 immune response in amammal comprising administering to the mammal an immunomodulatorycomposition in an amount sufficient to stimulate the Th1 immuneresponse, wherein the immunomodulatory composition comprises animmunomodulatory molecule, wherein said immunomodulatory moleculecomprises an antigen and is conjugated to an immunostimulatorypolynucleotide (ISS-PN), said ISS-PN comprising an immunostimulatorysequence (ISS) comprising the sequence 5′-cytosine, guanine-3′.
 30. Themethod of claim 29, wherein the ISS is at least six nucleotides inlength.
 31. The method of claim 29, wherein the ISS comprises thesequence 5′-Purine, Purine, C, G, Pyrimidine, Pyrimidine-3′.
 32. Themethod of claim 29, wherein the polynucleotide further comprises aphosphate backbone modification.
 33. The method of claim 29, wherein thecomposition further comprises an adjuvant.
 34. The method of claim 29,wherein the antigen is covalently conjugated to the polynucleotide. 35.The method of claim 29, wherein the antigen is non-covalently conjugatedto the polynucleotide.
 36. The method of claim 29, wherein the antigenis conjugated by a linker arm to the polynucleotide.